Non-insulin-dependent or type 2 diabetes mellitus is a metabolic disease characterized by an elevated blood glucose concentration that results from inadequate insulin action in insulin-sensitive tissues and from abnormal insulin secretion. In the USA, Europe and most westernized countries, type 2 diabetes affects ~6% of the population. It is estimated that by 2010, >220 million people worldwide will be affected by the disease, promoted by a dramatic increase in the incidence of obesity and a sedentary lifestyle. To achieve glucose control and prevent diabetic complications (retinopathy, cardiovascular disease, and nephropathy, among others), patients currently receive therapeutic agents that have limited efficacy and are associated with side- effects. Thus, there is a need to develop more efficient therapeutic agents. Our long-term goal is to identify gene targets to improve the treatment of type 2 diabetes. Current technologies to validate the role of genes in disease involve the development of animal models with null alleles for the target gene. The small interfering RNA (siRNA) technology has become the tool of choice to study gene function and validate targets because it allows carrying out studies in adult animals, making the study more relevant to the human condition. It also significantly reduces the time between functional studies and target validation. Gutless adenoviral vectors are optimal tools for gene delivery to the liver because they result in long-term expression of transgenes and negligible toxicity when administered at high doses. We have developed a gutless adenoviral vector system to express small hairpin RNA (shRNA) in liver. Higher than 75% gene silencing can be achieved, making this system a remarkable instrument for studies in this tissue. However, current limitations to using RNAi are the presence of toxic effects, which is a concern for its use as a research tool. The objective of the present proposal is to generate systems that use promoters with low transcriptional activity and inducible systems, so that the level of shRNA can be regulated by administration of a drug. We expect that the objective of this proposal will be achieved by pursuing the following specific aims: (i) Establish the level of gene downregulation that can be achieved using tissue-specific promoters; (ii) Identify cellular pathways affected by high-level shRNA expression in vivo; and (iii) Develop regulated transcription systems for synthesis of shRNA. We expect that the proposed research will provide the proof of concept that adenovirus-mediated shRNA delivery is a feasible strategy to generate long-term and inducible gene silencing. The system could be used as research tool for a variety of applications, which include not only diabetes, but also studies on liver-related diseases such as cardiovascular disease, hepatitis, and cancer. Public Health Relevance: The work proposed in this application will provide novel tools to identify drug targets for type 2 diabetes, a disease that affects approximately 6% of the US population. The data generated will provide information on basic aspects of siRNA biology, a promising therapeutic agent for the treatment of diseases such as diabetes, obesity, infectious diseases, and cancer.